The Lyme disease vaccine is based on OspA, a Borrelia burgdorferi lipoprotein, and immunity is contingent upon high levels of OspA antibodies. Toll-like receptors (TLRs) are important for the initiation of immune responses to pathogens and TLR2 recognizes bacterial lipoproteins, including OspA. Our preliminary data now demonstrate that TLR1 is also involved in recognizing OspA. Therefore defects in TLR-mediated signaling could result in ineffective lipoprotein-recognition and influence responsiveness to OspA-vaccination. In this proposal we will explore the association between TLR1, TLR2, and responsiveness to vaccination with OspA in mice and humans. We have now identified 7 persons with very low OspA antibody titers after vaccination. Macrophages from these individuals produced less TNF-a after stimulation with OspA - but not peptidoglycan - than controls; suggesting a defect in signaling that is partially associated with TLR2 (because peptidoglycan is also recognized by TLR2). In vitro transfection studies then demonstrated that dominant-negative TLR1 could inhibit TLR2-mediated OspA responsiveness, implying that TLR1 and TLR2 cooperate for lipoprotein recognition. This was further examined in Tlr2-/- mice, and Tlr1-/-mice that we have created. Tlr1-/- or Tlr2-/- mice made lower levels of OspA antibodies, after immunization with OspA, than controls. Macrophages from Tlr2-/- mice responded poorly to both OspA and peptidoglycan whereas cells from Tlr1-/- mice responded to peptidoglycan - but not OspA. These preliminary data suggest that TLR1 and TLR2 are required for OspA recognition, that the absence of TLR1 or TLR2 results in impair antibody responses to OspA immunization in mice, and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination. We will now fully characterize the response to OspA in Tlr1-/- or Tlr2-/- mice, and in humans that do not develop significant OspA antibodies following vaccination. These studies may provide an understanding of the molecular mechanisms for variability in antibody responses to the OspA vaccine, a first glimpse into TLR deficiencies in humans, and lead to new approaches to augment immune responses to OspA and other vaccines.